Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity in the female tract in a process known as capacitation. Initially, capacitation was defined using fertilization as end-point. However, a variety of evidences suggest that the functional changes occurring in the sperm during capacitation are not one event, but a combination of sequential and concomitant processes. Some of these processes occur as soon as the sperm are released from the epididymis, others are slower and are activated only after sperm incubation for a certain period of time in conditions that support the sperm ability to fertilize the egg. These slow events are associated with changes in the motility pattern (e.g. hyperactivation) and with the acquisition of the sperm capacity to undergo an agonist-stimulated acrosome reaction (AR). Although both, fast and slow events are regulated by HCO3- , activation of Adcy10, the atypical soluble adenylyl cyclase (also known as sAC) and the subsequent activation of a cAMP-dependent pathway, slower events are limited by the release of cholesterol from the sperm plasma membrane. Using the mouse as an experimental model, we have shown that these last events are associated with a protein kinase A (PKA)-dependent increase in protein tyrosine (tyr) phosphorylation. We have also demonstrated that the increase in tyr phosphorylation as well as capacitation was regulated by the presence of cholesterol acceptors such as Bovine Serum Albumin (BSA) in the capacitation media. During the first cycle of this proposal we have discovered that although necessary, the classical linear pathway involving HCO3-/SAC/PKA is not sufficient to elicit phosphorylation of downstream targets. On top of PKA activation, another pathway leading to downregulation of ser/thr phosphatases is also necessary. This second pathway is essential to modulate the PKA pathway and for the activation of other PKA- independent phosphorylation pathways needed for sperm capacitation. The hypotheses underlying this proposal postulate that capacitation results from the combined action of two pathways, one regulated by cholesterol acceptors (e.g. BSA) inducing a cSrc family kinase (SFK)-induced downregulation of ser/thr phosphatase(s) and the other regulated by HCO3- and Ca2+ and mediated by the activation of sAC and PKA. The objective of this proposal is to understand how these two pathways are integrated during capacitation. PUBLIC HEALTH RELEVANCE: Difficulties in earlier efforts to fertilize mammalian eggs in vitro were due mainly to a lack of comprehension of sperm physiology. This proposal is aimed to understand the molecular basis of sperm capacitation with emphasis in the regulation of signaling pathways involving protein phosphorylation in sperm. Accomplishment of these goals will lead to novel insights in clinical diagnosis of male infertility and provide tools for evaluating pharmacological contraceptive targets.